Micro-computed tomography analysis of changes in the periodontal ligament and alveolar bone proper induced by occlusal hypofunction of rat molars
大鼠磨牙咬合功能减退诱导的牙周韧带和牙槽骨变化的显微计算机断层扫描分析

Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
日本东京医科齿科大学研究生院口腔健康科学部口腔面发育与功能系口腔正畸科学

Objective: To three-dimensionally elucidate the effects of occlusal hypofunction on the periodontal ligament and alveolar bone proper of rat molars by microcomputed tomography (micro-CT). Methods: Occlusal function in the molar area was restricted by attaching an anterior bite plate on the maxillary incisors and a metal cap on the mandibular incisors of 5-week-old male Wistar rats for 1 week. The periodontal ligament space and alveolar bone proper around roots of the mandibular first molar were assessed by histology and micro-CT. Results: The periodontal ligament space was narrower and the alveolar bone proper was sparser and less continuous in the hypofunction group than in the control group. Further, both the volume of the periodontal ligament and the volumetric ratio of the alveolar bone proper to the total tissue in the region of interest were significantly lower in the hypofunction group ( $p<0.05$$p<0.05$p < 0.05p<0.05 ). Conclusions: Occlusal hypofunction induces atrophic changes in the periodontal ligament and alveolar bone proper of rat molars.
目的： 通过显微计算机断层扫描 （micro-CT） 三维阐明咬合功能减退对大鼠磨牙牙周韧带和牙槽骨的影响。方法： 通过在 5 周龄雄性 Wistar 大鼠的上颌切牙上连接前咬合板和在下颌切牙上连接金属帽 1 周来限制磨牙区域的咬合功能。通过组织学和显微 CT 评估下颌第一磨牙根周围的牙周韧带间隙和牙槽骨。结果： 与对照组相比，功能减退组的牙周韧带间隙更窄，牙槽骨本身更稀疏且连续性较差。此外，在功能减退组中，牙周韧带的体积和牙槽骨占感兴趣区域总组织的体积比均显着降低 （ $p<0.05$$p<0.05$p < 0.05p<0.05 ）。结论： 咬合功能减退会引起大鼠磨牙牙周韧带和牙槽骨的萎缩变化。
[Korean J Orthod 2014;44(5):263-267]
[韩国 J Orthod 2014;44(5):263-267]
Key words: Three dimensional scanner, Computed tomography, Histology, Bone biology, Occlusal stimuli, Micro-computed tomography
关键词 ： 三维扫描仪， 计算机断层扫描， 组织学， 骨生物学， 咬合刺激， 显微计算机断层扫描

Received November 25, 2013; Revised February 27, 2014; Accepted March 18, 2014.
接收日期：2013 年 11 月 25 日;2014 年 2 月 27 日修订;2014 年 3 月 18 日接受。
Corresponding author: Yasuhiro Shimizu.
通讯作者：Yasuhiro Shimizu。
Clinical Fellow, Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
东京医科齿科大学研究生院口腔健康科学部口腔面发育与功能系正畸科学临床研究员，1-5-45 Yushima， Bunkyo-ku， Tokyo 113-8549， Japan。
Tel +81-3-5803-5528 e-mail y.shimizu.orts@tmd.ac.jp
电话 +81-3-5803-5528 电子邮件 y.shimizu.orts@tmd.ac.jp
*This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI; grant number 24890062) from the Japan Society for the Promotion of Science (JSPS).
*这项工作得到了日本科学振兴会 （JSPS） 的科学研究资助 （KAKENHI;资助号 24890062） 的支持。

The authors report no commercial, proprietary, or financial interest in the products or companies described in this article.
作者报告称，本文中描述的产品或公司没有商业、专有或财务利益。
© 2014 The Korean Association of Orthodontists.
© 2014 年韩国正畸医师协会。

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
这是一篇根据知识共享署名非商业许可证 （http://creativecommons.org/licenses/by-nc/3.0） 条款分发的开放获取文章，该许可证允许在任何媒体上不受限制地非商业使用、分发和复制，前提是正确引用原始作品。

The compact bone lining the tooth socket, where periodontal ligament (PDL) fibers attach, is known as bundle bone or alveolar bone proper. It is easily identifiable in radiographs and histological sections. The PDL and alveolar bone proper have a close relationship: the PDL provides progenitor cells for bone formation and remodeling. ${}^{1-4}$${}^{1-4}$^(1-4){ }^{1-4}
牙槽内衬的致密骨是牙周韧带 （PDL） 纤维附着的地方，被称为束骨或牙槽骨。在 X 光片和组织学切片中很容易识别。PDL 和牙槽骨本身有着密切的关系：PDL 为骨骼形成和重塑提供祖细胞。 ${}^{1-4}$${}^{1-4}$^(1-4){ }^{1-4}
Occlusal hypofunction leads to atrophic changes in the periodontium. ${}^{5-12}$${}^{5-12}$^(5-12){ }^{5-12} These changes include narrowing of the PDL space, vascular constriction, and deformation of the mechanoreceptor structure (Ruffini endings). Therefore, occlusal stimuli are essential for maintaining the structural integrity of the periodontium. ${}^{5-7,11,12}$${}^{5-7,11,12}$^(5-7,11,12){ }^{5-7,11,12}
咬合功能减退导致牙周萎缩性变化。 ${}^{5-12}$${}^{5-12}$^(5-12){ }^{5-12} 这些变化包括 PDL 空间变窄、血管收缩和机械感受器结构变形（Ruffini 末端）。因此，咬合刺激对于维持牙周组织的结构完整性至关重要。 ${}^{5-7,11,12}$${}^{5-7,11,12}$^(5-7,11,12){ }^{5-7,11,12}
Previous histological studies have shown regressive changes in the periodontium of rat molars after loss of occlusal stimuli, ${}^{7,11,12}$${}^{7,11,12}$^(7,11,12){ }^{7,11,12} but three-dimensional morphometric analysis has not yet been reported. In this study, we aimed to three-dimensionally elucidate the effects of occlusal hypofunction on the PDL and alveolar bone proper of rat molars by micro-computed tomography (micro-CT).
以前的组织学研究表明，咬合刺激丧失后大鼠磨牙牙周的回归变化， ${}^{7,11,12}$${}^{7,11,12}$^(7,11,12){ }^{7,11,12} 但尚未报道三维形态学分析。在这项研究中，我们旨在通过显微计算机断层扫描 （micro-CT） 三维阐明咬合功能减退对大鼠磨牙 PDL 和牙槽骨本体的影响。

Five-week-old male Wistar rats $(\mathrm{n}=18$$(\mathrm{n}=18$(n=18(\mathrm{n}=18 ) were randomly divided into two equal groups. The control group was not treated. To restrict occlusal function in the hypofunction group, an anterior bite plate was attached on the maxillary incisors and a metal cap constructed from orthodontic band material ( $4.6\times 0.13\mathrm{mm}$$4.6\times 0.13\mathrm{mm}$4.6 xx0.13mm4.6 \times 0.13 \mathrm{~mm}; Rocky Mountain Morita Corp., Tokyo, Japan) was attached on the mandibular incisors by using photopolymerizing composite resin (Clearfil Liner Bond 2; Kuraray Dental, Okayama, Japan) (Figure 1A). ${}^{8-10}$${}^{8-10}$^(8-10){ }^{8-10} Body weight was monitored daily. ${}^{8-10}$${}^{8-10}$^(8-10){ }^{8-10} One week later, the animals were sacrificed by cervical dislocation under diethyl ether anesthesia (Wako Pure Chemical Ind. Ltd., Osaka, Japan). ${}^{8,10}$${}^{8,10}$^(8,10){ }^{8,10} The experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Tokyo Medical and Dental University (approval number #0130279A).
将 5 周龄雄性 Wistar 大鼠 $(\mathrm{n}=18$$(\mathrm{n}=18$(n=18(\mathrm{n}=18 随机分为 2 组。对照组未接受治疗。为了限制功能减退组的咬合功能，在上颌切牙上连接了前咬合板，并用正畸带材料制成的金属帽 （ $4.6\times 0.13\mathrm{mm}$$4.6\times 0.13\mathrm{mm}$4.6 xx0.13mm4.6 \times 0.13 \mathrm{~mm} ;Rocky Mountain Morita Corp.，日本东京）通过使用光聚合复合树脂（Clearfil Liner Bond 2;日本冈山市 Kuraray Dental）（图 1A）。 ${}^{8-10}$${}^{8-10}$^(8-10){ }^{8-10} 每天监测体重。 ${}^{8-10}$${}^{8-10}$^(8-10){ }^{8-10} 一周后，在乙醚麻醉下通过宫颈脱位处死动物 （Wako Pure Chemical Ind. Ltd.， Osaka， Japan）。 ${}^{8,10}$${}^{8,10}$^(8,10){ }^{8,10} 实验程序由东京医科齿科大学机构动物护理和使用委员会（批准号 #0130279A）审查和批准。

The mandibles were dissected and most soft tissue was removed. The specimens were immediately immersed
解剖下颌骨并切除大部分软组织。立即将标本浸入水中

Figure 1. A, The experimental model; B, histological observational area (rectangular area); C, representative hematoxylin and eosin-stained sections (Scale $=250\mu \mathrm{m}$$=250\mu \mathrm{m}$=250 mum=250 \mu \mathrm{~m} ). M, Mesial; D, distal; PL, periodontal ligament; AP, alveolar bone proper; AB, inter-radicular alveolar bone.
图 1.A， 实验模型;B， 组织学观察区 （矩形区）;C，代表性苏木精和伊红染色切片（量表 $=250\mu \mathrm{m}$$=250\mu \mathrm{m}$=250 mum=250 \mu \mathrm{~m} ）。M， 近中;D，远端;PL， 牙周韧带;AP，牙槽骨本身;AB，根间牙槽骨。
in 10% buffered formalin (Wako Pure Chemical Ind. Ltd.; pH 7.0 ) at $4^{\circ }\mathrm{C}$$4^{\circ }\mathrm{C}$4^(@)C4^{\circ} \mathrm{C} overnight and decalcified in $10\mathrm{\%}$$10\mathrm{\%}$10%10 \% ethylenediaminetetraacetic acid (EDTA) at $4^{\circ }\mathrm{C}$$4^{\circ }\mathrm{C}$4^(@)C4^{\circ} \mathrm{C} for 6 weeks. They were embedded in paraffin and cut into $5-\mu \mathrm{m}$$5-\mu \mathrm{m}$5-mum5-\mu \mathrm{m}-thick sagittal sections parallel to the long axis of the roots of the mandibular first molar. ${}^{8,10}$${}^{8,10}$^(8,10){ }^{8,10} The sections were finally mounted on glass slides coated with poly-L-lysine (Matsunami Glass Ind. Ltd., Osaka, Japan) and stained with hematoxylin and eosin for histomorphometric examination. The observational area included the dental pulp, PDL, alveolar bone proper, and inter-radicular alveolar bone (Figure 1B).
在 10% 缓冲福尔马林 （Wako Pure Chemical Ind. Ltd.;pH 7.0） 中 $4^{\circ }\mathrm{C}$$4^{\circ }\mathrm{C}$4^(@)C4^{\circ} \mathrm{C} 过夜，并在乙二胺四乙酸 （EDTA） $4^{\circ }\mathrm{C}$$4^{\circ }\mathrm{C}$4^(@)C4^{\circ} \mathrm{C} 中 $10\mathrm{\%}$$10\mathrm{\%}$10%10 \% 脱钙 6 周。它们被嵌入石蜡中，并切成 $5-\mu \mathrm{m}$$5-\mu \mathrm{m}$5-mum5-\mu \mathrm{m} 平行于下颌第一磨牙根部长轴的厚矢状面。 ${}^{8,10}$${}^{8,10}$^(8,10){ }^{8,10} 最后将切片安装在涂有聚-L-赖氨酸（Matsunami Glass Ind. Ltd.，日本大阪）的载玻片上，并用苏木精和伊红染色以进行组织形态学检查。观察区域包括牙髓、PDL、牙槽骨本身和根间牙槽骨（图1B）。

The histological observational area was imaged by using a desktop X-ray micro-CT system (SMX90CT; Shimadzu Corp., Kyoto, Japan) with a scanning resolution of $20\mu \mathrm{m}$$20\mu \mathrm{m}$20 mum20 \mu \mathrm{~m}. First, the mesial and distal roots of the mandibular first molar were manually located at $20\mu \mathrm{m}$$20\mu \mathrm{m}$20 mum20 \mu \mathrm{~m} intervals on individual images. The selected images were compiled into a three-dimensional image by using image-analysis software (TRI/3-D Bon; Ratoc System Engineering Co. Ltd., Tokyo, Japan). The region of interest was enlarged to a $40\mu \mathrm{m}$$40\mu \mathrm{m}$40 mum40 \mu \mathrm{~m} radius around
使用台式 X 射线显微 CT 系统 （SMX90CT;Shimadzu Corp.， Kyoto， Japan），扫描分辨率为 $20\mu \mathrm{m}$$20\mu \mathrm{m}$20 mum20 \mu \mathrm{~m} 。首先，在单个图像上间隔手动定位 $20\mu \mathrm{m}$$20\mu \mathrm{m}$20 mum20 \mu \mathrm{~m} 下颌第一磨牙的近中根和远中根。使用图像分析软件 （TRI/3-D Bon;Ratoc System Engineering Co. Ltd.，日本东京）。感兴趣区域扩大到 $40\mu \mathrm{m}$$40\mu \mathrm{m}$40 mum40 \mu \mathrm{~m} 周围

A
Periodontal ligament space of mesial root
近中根牙周韧带间隙

Control group 控制组

Hypofunction group 功能减退组
B

Figure 2. A, Three-dimensional reconstructed images of the periodontal ligament (PDL) space (Scale $=1\mathrm{mm}$$=1\mathrm{mm}$=1mm=1 \mathrm{~mm} ); B, comparison of tissue volume (TV) around the mesial root of the mandibular first molar.
图 2.A， 牙周韧带 （PDL） 空间的三维重建图像 （Scale $=1\mathrm{mm}$$=1\mathrm{mm}$=1mm=1 \mathrm{~mm} ）;B，下颌第一磨牙近中根周围的组织体积 （TV） 比较。
${}^{\ast \ast \ast }p<0.001$${}^{\ast \ast \ast }p<0.001$^(******)p < 0.001{ }^{* * *} p<0.001 by the Mann-Whitney U-test. M, Mesial; D, distal.
${}^{\ast \ast \ast }p<0.001$${}^{\ast \ast \ast }p<0.001$^(******)p < 0.001{ }^{* * *} p<0.001 由 Mann-Whitney U 检验。M， 近中;D， 远端。
the roots. ${}^{8-10}$${}^{8-10}$^(8-10){ }^{8-10} The software was then used for data quantification. Tissue volume (TV) was defined as the volume of tissue in the enlarged region of interest. The bone-to-tissue volume (BV/TV) ratio was calculated as the volumetric ratio of bone to total tissue in the enlarged region of interest. Both parameters were evaluated according to the software instructions.
根。 ${}^{8-10}$${}^{8-10}$^(8-10){ }^{8-10} 然后使用该软件进行数据量化。组织体积 （TV） 定义为感兴趣区域内的组织体积。骨组织体积 （BV/TV） 比值计算为扩大的感兴趣区域中骨与总组织的体积比。根据软件说明评估两个参数。

Statistical calculations were performed by using StatView 5.0 software (SAS Institute, Cary, NC, USA). The Mann-Whitney U-test was used for all comparisons at the significance level of $p<0.05$$p<0.05$p < 0.05p<0.05.
使用 StatView 5.0 软件 （SAS Institute， Cary， NC， USA） 进行统计计算。Mann-Whitney U 检验用于显著性水平 的所有 $p<0.05$$p<0.05$p < 0.05p<0.05 比较。

No significant differences in body weight were noted between the groups at the time of killing.
杀戮时两组之间的体重没有显著差异。

Histologically, the PDL space in the hypofunction group was narrower than that in the control group (Figure 1C). Furthermore, the alveolar bone proper showed less continuity in the hypofunction group (Figure 1C).
组织学上，功能减退组的 PDL 空间比对照组窄（图 1C）。此外，功能减退组的牙槽骨本身的连续性较差（图 1C）。
Figure 2A shows that the PDL space around the mesial
图 2A 显示近中

A
Periodontal ligament space of distal root
远端根部牙周韧带间隙

B

Figure 3. A , Three-dimensional reconstructed images of the periodontal ligament ( PDL ) space (Scale $=1\mathrm{mm}$$=1\mathrm{mm}$=1mm=1 \mathrm{~mm} ); B, comparison of tissue volume (TV) around the distal root of the mandibular first molar.
图 3.A，牙周韧带 （PDL） 空间的三维重建图像 （Scale $=1\mathrm{mm}$$=1\mathrm{mm}$=1mm=1 \mathrm{~mm} ）;B，下颌第一磨牙远端根部周围的组织体积 （TV） 比较。
${}^{\ast \ast \ast }p<0.001$${}^{\ast \ast \ast }p<0.001$^(******)p < 0.001{ }^{* * *} p<0.001 by the Mann-Whitney U-test.
${}^{\ast \ast \ast }p<0.001$${}^{\ast \ast \ast }p<0.001$^(******)p < 0.001{ }^{* * *} p<0.001 由 Mann-Whitney U 检验。
M, Mesial; D, distal. M， 近中;D， 远端。